Detection of hypertension using immunoreactive metabolic products

ABSTRACT

A method to assess hypertension by measuring the amount of free and conjugated hydroxyeicosatrienoic acids (DHETS) and metabolites of DHETs, which are metabolites of arachidonic acid (AA) epoxygenases and epoxide hydrolases, in a biological sample which contains the DHETs (using any methods including GC/MS or ELISA) is disclosed. The method further included determining the amount of molecules containing a DHET-specific epitope immunoreactive with antibodies produced against DHETs present in the sample. This amount is compared with a control sample(s). Hypertension is determined through the comparison wherein the amount of increase of free and conjugated DHETs and metabolites of DHETs in the sample isolated from an organism. The present invention also provides a method to assess catalytic activity of AA epoxygenases using immunoassays by measuring the amounts of NADPH-independent epoxyeicosatrienoic acids (EETs) and total (NADPH-dependent+independent) EETs. The present invention also provides a method to decrease hepatic AA epoxygenase expression including 2C23 by treatment of rats with a glucocorticoid including dexamethasone.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a conversion of U.S. Provisional Patent Application Ser. No., 60/136,475, filed May 28, 1999, claiming benefit under 35 U.S.C. §119 (e), and which is incorporated herein by reference.

GOVERNMENT SUPPORT

Research in this application was supported in part by a contract from National Institute of Environmental Health Sciences (ES 85430). The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a method to analyze arachidonic acid (AA)-derived products which are immunoreactive with antibodies produced against hydroxyeicosatrienoic acids (DHETs). More specifically, the present invention relates to a method which can be used to facilitate investigations of the physiological and pathophysiological roles of the metabolic products of arachidonic acid epoxygenases and epoxide hydrolases. The present invention also relates to a method to assess catalytic activity of AA epoxygenases using and a method to decrease hepatic AA epoxygenase expression by treatment with glucocorticoids.

2. Description of Related Art

AA is a component of cellular membranes and plays a critical role as a mediator of cell and organ function through its metabolic cascade. The AA cascade includes prostaglandin synthases, lipoxygenases, and cytochromes P450 (CYPs). The CYP pathway is composed of lipoxygenases-like (allylic oxidation), ω/ω-1 oxygenases and epoxygenases (olefin epoxidation), which metabolize AA to produce 5-, 8-, 9-, 11-, 12-, and 15-hydroxyeicosatetraenoic acids (HETEs), 16- to 20-hydroxyeicosatetraenoic acids (OH-AAs), and 5,6-, 8,9-, 11,12- and 14,15-epoxyeicosatrienoic acids (EETs), respectively (1). Epoxide hydrolases hydrolyze biologically active EETs to their corresponding dihydroxyeicosatrienoic acids (DHETs).

CYPs which have been identified to be AA epoxygenases are CYP2C11, CYP2C23, CYP2B1, and CYP2B2 expressed in rats (1), CYP2C1 and CYP2C2 expressed in rabbits (2,3), and CYP2C8 and CYP2C9/2C10 expressed in human (4). Recently, CYP2J, a novel CYP subfamily which is abundantly expressed in extrahepatic tissues, has been found in humans (CYP2J2) (5), rats (CYP2J3) (6) and rabbits (CYP2J1) (7).

EETs are synthesized in many tissues including liver, brain, eye, adrenal gland, blood vessels, and kidney, and circulate in blood. EETs are excreted in urine (8-10). Recently, it has been reported that EETs and DHETs are also synthesized in the lung, heart and gastrointestinal tract (5,7,11).

EETs have potent vasoactive properties (vasodilator or vasoconstrictor), increase cytosolic Ca⁺ concentration and renal Na⁺ transport, and stimulate hormone release in many tissues including insulin and glucagon from pancreatic islet cells, growth horrnone, oxytocin and vasopressin from the pituitary gland, and catecholamines from adrenal gland (9). Recently, it is reported that 14,15-EET functions as a second messenger in epidermal growth factor-mediated signaling pathway (12).

Urinary excretion of Na⁺, EETs and DHETs decreased after inhibition of AA epoxygenase activity by treating rats with clotrimazole, which induced salt-sensitive and clotrimazole-dependent hypertension. A salt-sensitive phenotype of the Dahl rat was associated with a lack of increases in renal AA hypoxygenases after intake of a high salt diet (10,13). A spontaneous hypertensive rat (SHR) study indicated that ω/ω-1 hydroxylase activity of kidney microsomes was significantly higher than that of normotensive Witstar Kyoto rats (WKY) whereas AA epoxygenase activity (EETs+DHETs) showed no difference between two strains at any age group tested (14). Urine samples were not tested. Thus it is generally concluded that the developmental phase of hypertension was linked to increases in the activity of kidney microsomal ω/ω-1 hydroxylase. Indeed, recently the gene coding for CYP4A2 (ω/ω-1 hydroxylase) was found to be preferentially expressed in SHR (14,15).

So far, levels of urinary EETs or DHETs of SHR have not been measured or compared with those of WKY. Thus, our findings that DHET levels in urine specimens obtained from SHR is ˜56-fold higher than those of WKY and existence of free and conjugated DHETs and metabolites of DHETs in the SHR urine specimens were surprising. Our result strongly suggest that epoxide hydrolases expressed in kidney play a critical role in hypertension. Thus, measurement of total DHET levels in urine provides better correlation of AA epoxygenase-epoxide hydrolase activities with hypertension.

Levels of EETs in biological specimens, thus far, were measured after chemical hydrolysis to DHETs by high-performance liquid chromatography (HPLC) followed by negative-ion chemical ionization/gas chromatography/mass spectrometry (NICI/GC/MS). This method is not suitable for clinical routines because a) sample preparation for GC/MS is long and tedious, b) The method assesses only free EETs, EETs after chemical hydrolysis to DHETs, or DHETs and c) it is not practical to measure all the possible EET or DHET metabolites using GC/MS. EET levels in human urine samples and human urine specimens during pregnancy were assayed. However, the EET levels were measured after chemical hydrolysis to DHETs by HPLC/GC/MS (16,17). So, although the data is shown to be DHET, it is only measuring primarily EETs converted to DHET.

Recently free EETs (after chemical hydration to DHETs) were measured by using fluoroimmunoassay (FIA) and radioimmunoassay (RIA) to measure EET release from cells (18). However, again the EET levels of the cell medium extract were measured after chemical hydrolysis of the EETs to DHETs. No prior art reference directs one skilled in the art to the important relationship between the total (free and conjugated) metabolites of AA epoxygenases and epoxide hydrolases and hypertension, nor their measurement in urine samples.

SUMMARY OF THE INVENTION

According to the present invention, a method to assess hypertension mediated by AA epoxygenase and epoxide hydrolase by measuring the amount of metabolites of epoxygenases and epoxide hydrolases (DHETs) in a biological sample which contains the DHETs (using any methods including GC/MS or ELISA). The method further included determining the amount of DHET-specific epitopes immunoreactive with antibodies produced against DHETs present in the sample. This amount is compared with a control sample(s). The hypertension mediated by AA epoxygenase and epoxide hydrolase is determined through the comparison wherein the amount of increase in the free and conjugated DHETs and metabolites of DHETs in the sample isolated from an organism. The present invention also provides an immunoassay to assess catalytic activity of AA epoxygenases by subtracting the amounts of NADPH-independent EETs from total (NADPH-dependent+independent) EETs after incubation of the enzyme with NADPH. The present invention also provides a method to decrease hepatic AA epoxygenase expression by treatment with a glucocorticoid including dexamethasone.

DESCRIPTION OF THE DRAWINGS

Other advantages of the present invention will be readily appreciated as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings wherein:

FIG. 1 is a graph showing levels of 14,15-DHET in urine samples obtained from spontaneously hypertensive rats (SHR) and normotensive Vistar Kyoto rats (WKY);

FIGS. 2A-B are graphs showing cross-reactivity of anti-14,15-and 8,9-dihydroxyeicosatrienoic acid (DHET) with DHET conjugated bovine serum albumin (BSA); cross-reactivity of preimmune sera or antibodies produced against 14,15-DHET (Panel A) or 8,9-DHET (Panel B) conjugated to kyehole limpet hemocyanin (KLH) being determined by ELISA; one μg of DHET conjugated BSA per well was coated on plates; whereas preimmune did not show cross-reactivity, immune sera showed strong cross-reactivity with DHET conjugated BSA;

FIG. 3 is a graph showing assessment of arachidonic acid (AA) epoxygenase activity using a competitive ELISA against 14,15-dihydroxyeicosatrienoic acid (DHET). NADPH-dependent or independent 14,15-epoxyeicosatrienoic acid (EET) formation was measured by 14,15-DHET ELISA after chemical hydrolysis to DHET.

FIG. 4 shows a decrease of hepatic CYP2C23 after treatment of rats with dexamethasone; lanes 1 to 3, hepatic microsomes obtained from male rat; lane 4 to 6, microsomes obtained from female rats; lanes 1 and 4, microsomes obtained from untreated rats; lanes 2 and 5, microsomes obtained from rats after 4 days of corn oil treatment (2 ml/kg/day, i.p.); lanes 3 and 6, microsomes obtained from rats after 4 days of dexamethasone treatment (10 mg/kg/day, i.p.);

FIG. 5 is a graph showing the sensitivity of IgG produced against 14,15-dihydroxyeicosatrienoic acid (DHET): a typical standard curve for 14,15-DHET in a competitive ELISA;

FIG. 6 shows the specificity of anti-14,15-dihydroxyeicosatrienoic acid (DHET) in slot blot analysis;

FIGS. 7A-B show the specificity of anti-8,9-dihydroxyeicosatrienoic acid (DHET) is slot blot analyses without (Panel A) and with (Panel B) immunoaffinity column purification; and

FIG. 8 is a graph showing retention of 14,15-dihydroxyeicosatrienoic acid (DHET) in an immunoaffinity column.

DETAILED DESCRIPTION OF THE INVENTION

The present invention generally provides, a method to assess hypertension mediated by AA epoxygenase and epoxide hydrolase by measuring the amount of free and conjugated DHETs and metabolites of DHETs, which are metabolites of AA epoxygenases and epoxide hydrolases, in a biological sample which contains the DHETs. The method further includes determining the amount of molecules containing DHET-specific epitopes immunoreactive with antibodies produced against DHETs present in the sample. This amount is compared with a healthy control sample(s). In general, a panel of healthy control samples is used that are within the normal range. The normal range is established as known in the art and is established for each assay method being utilized, e.g. GC/MS and immunoassays. The hypertension is determined through the comparison wherein the amount of the DHETs is increased in the sample isolated from an organism hypertensive compared to controls.

By assessing hypertension, it is meant that the present inventive assay is capable of being an indication of hypertension. In combination with conventional detection methods, the assay indicates a relationship between hypertension and elevated enzyme activity in the AA cascade. Specifically, the activity of the two enzymes, the epoxygenase and hydrolase which produce DHETs, are unexpectedly correlated with the hypertension.

The biological sample can be selected from biological fluids which contain the DHETs and can include, but are not limited to, plasma, urine, cerebrospinal fluids, bile and joint fluids. Urine is the preferred sample.

It has been previously shown that AA epoxygenase activities (EETs+DHETs) of kidney cortical microsomes obtained from spontaneously hypertensive rats (SHR) were not different from those of normotensive Wistar Kyoto rats (WKY) (14). Applicants determined DHET levels in urine samples obtained from SHR and compared with those of WKY and found that contrary to the results obtained with kidney cortical microsome study of those rats, urinary DHET levels of SHR were ˜56-fold higher than those of WKY (Table 1; FIG. 1). Thus, measurement of urinary DHETs provides better (more specific and more sensitive) assessment of hypertension mediated by AA epoxygenase and epoxide hydrolase (Tables 1 and 2; FIG. 1).

The preferred assay of the present invention, as discussed in detail in the Experiment section herein, is sensitive to both conjugate-free and conjugated forms of DHETs. Recovery rate of ³H 14,15- and ³H8,9-DHETs spiked in human urine after liquid phase extraction with ethyl acetate was ˜100% (Table 3). However, 14,15-DHET levels in urine samples obtained from SHR decreased to the levels lower than 40% after ethylacetate extraction (Tables 1 and 2; FIG. 1). This result demonstrated that 14,15-DHET exists in the urine as free (ethyl acetate extractable) and conjugated (ethyl acetate unextractable) forms and antibodies produced in accordance with the present invention against 14,15-DHETs recognize both free and conjugated forms. Cross-reactivity of the antibodies with the conjugated form of 14,15- and 8,9-DHETs was further demonstrated by ELISAs using 14,15- and 8,9-DHETs conjugated to bovine serum albumin (FIG. 2, Panels A and B). The antisera recognized the conjugated forms whereas preimmune sera showed only a basal level cross-reactivity with the conjugated forms.

It was surprising that antibodies produced against DHETs

a) recognize free (ethyl acetate extractable) and conjugated (ethyl acetate unextractable) forms of DHETs (Tables 1 and 2; FIG. 1);

b) are useful for detection of hypertension related to AA oxygenase and epoxyde hydrolase activities and

c) useful for measurement of AA oxygenase (FIG. 3) and epoxide hydrolase activities.

It was unexpected to find that NADPH-independent (AA-epoxygenase-independent) EET formation with hepatic microsomes (FIG. 3). AA-epoxygenase activity analysis using ¹⁴C AA (Table 4) measures only AA-epoxygenase-dependent EET formation whereas assessment of EETs using immunoassays measures both AA epoxygenase-dependent and independent EET formation. Dexamethasone treatment increased AA epoxygenase-independent EET formation activity in liver microsomes (FIG. 3). Subtracting the amounts of NADPH-independent EETs from total (NADPH-dependent+NADPH-independent) EETs after incubation of the enzyme with NADPH provides better assessment of catalytic activity of AA epoxygenases.

Epoxide hydrolase activity also can be measured by the ELISAs against 14,15-DHET because anti-DHET does not recognize 14,15-EET.

It has been previously shown that CYP2C23 is one of AA epoxygenases expressed in rats. So far, CYP2C11, CYP2C23, CYP2B1, CYP2B2, and CYP2J3 are identified as AA epoxygenases expressed in rats (1). Dexamethasone treatment substantially decreased hepatic AA epoxygenase activity which coincided with decrease in CYP2C23 protein levels (FIG. 4). This unexpected result showed that 2C23 is a predominant AA epoxygenase isoform in the rat liver and hepatic AA epoxygenase expression is lowered by treatment of rats with chemicals such as glucocorticoids including dexamethasone.

In general the quantification of the sample is done utilizing an immunoassay as described in the Examples herein. However alternative immunoassays or GC/MS can be used as the assay in accordance with the present invention. Most of the techniques used in performing immunoassays are widely practiced in the art, and most practitioners are familiar with the standard resource materials which describe specific conditions and procedures. However, for convenience, the following paragraph may serve as a guideline.

In general, ELISAs are the preferred immunoassays employed to assess the amount of EETs and DHETs in a specimen. ELISA assays are well known to those skilled in the art. Both polyclonal and monoclonal antibodies can be used in the assays. Where appropriate other immunoassays, such as radioimmunoassays (RIAs) or fluoroimmunoassays (FIAs) can be used as are known to those in the art. Available immunoassays are extensively described in the patent and scientific literature. See, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521 and may be adapted to be used the is method of the present invention.

The free and conjugated forms of the EETs and DHETs are measured utilizing the immunoassay as set forth for example in the Examples herein with an antibody which recognized both forms. Alternatively, antibodies can be utilized which are specific for each form. Such antibodies can be produced as described herein and tested as set forth in Example 1.

Most of the techniques used to produce antibodies are widely practiced in the art, and most practitioners are familiar with the standard resource materials which describe specific conditions and procedures. However, for convenience, the following paragraphs may serve as a guideline.

Antibody production: Antibodies (immunoglobulins) may be either monoclonal or polyclonal and are raised against the immunogen. Such immunogens can be used to produce antibodies by standard antibody production technology well known to those skilled in the art as described generally in Harlow and Lane, Antibodies: A laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988 and Borrebaeck, Antibody Engineering—A practical Guide, W. H. Freeman and Co., 1992. Antibody fragments may also be prepared from the antibodies and include Fab, F(ab′)², and Fv by methods known to those skilled in the art.

For producing polyclonal antibodies a host, such as a rabbit or goat, is immunized with the immunogen, generally together with an adjuvant and, if necessary, coupled to a carrier: antibodies to the immunogen are collected from the sera. Further, the polyclonal antibody can be absorbed such that it is monospecific. That is, the sera can be absorbed against related immunogens, e.g. the free and conjugated forms of EETs and DHETs, so that no cross-reactive antibodies remain in the sera thereby rendering it monospecific. Testing for this specificity can be undertaken as described in Example 1.

For producing monoclonal antibodies the technique involves hyperimmunization of an appropriate donor with the immunogen or immunogen fragment, generally a mouse, and isolation of splenic antibody producing cells. These cells are fused to a cell having immortality, such as a myeloma cell, to provide a fused cell hybrid which has immortality and secretes the required antibody. The cells are then cultured, in bulk, and the monoclonal antibodies harvested from the culture media for use.

For producing recombinant antibody (19) (20) (21), messenger RNAs from antibody producing B-lymphocytes of animals, or hybridoma are reversetranscribed to obtain complimentary DNAs (cDNAs). Antibody cDNA, which can be full or partial length, is amplified and cloned into a phage or a plasmid. The cDNA can be a partial length of heavy and light chain CDNA, separated or connected by a linker. The antibody, or antibody fragment, is expressed using a suitable expression system to obtain recombinant antibody.

The antibody or antibody fragment can be bound to a solid support S substrate or conjugated with a detectable moiety or be both bound and conjugated as is well known in the art to be used in the immunoassay. (For a general discussion of conjugation of fluorescent or enzymatic moieties (22). The binding of antibodies to a solid support substrate is also well known in the art (22) (24). The detectable moieties contemplated with the present invention can include ferritin, alkaline phosphatase, b-galactosidase, peroxidase, urease, fluorescein, rhodamine, tritium, ¹⁴C and iodination as needed for the immunoassay.

The methods used with and the utility of the present invention can be shown by the following non-limiting examples and acompanying figures.

EXAMPLES

Materials and Methods

Materials

EETs and DHETs and ³H-labeled 14,15- and 8,9-DHETs (higher than 98% pure by HPLC and GC/MS) were provided by Dr. Jorge Capdevila's laboratory. Antibodies against CYP3A and CYP2C23 were obtained from Detroit R&D and Dr. Capdevila's laboratory, respectively. Horseradish peroxidase-conjugated donkey antigoat or goat anti-rabbit immunoglobulin G (IgG) and alkaline phosphatase-conjugated donkey anti-rabbit IgG were purchased from Jakson immunoResearch Laboratories, Inc. (West Grove, Pa.). 15(S)HETE, 5(s)15(S)DiHETE, arachidonic acid, Thromboxane B₂, PGE₂, PGF_(2α), 6-keto-PGF_(1α) were obtained from Biomol Research Lab (Plymouth Meeting, Pa.). Reactigel used to cross-link anti-14,15-DHET was purchased from Pierce (Rockford, Ill.). Other reagents were obtained from Sigma Chemical Co.

Antibody Production

Synthetic 14,15- or 8,9-DHETs were coupled to KLH using dicyclohexylcarbodiimide as previously described (25). The conjugate was used to immunize a goat and antibody titers were determined by ELISA using DHET-conjugated bovine serum albumin (BSA).

Purification of IqG Fraction of Antisera

IgG fractions of antibody prepared against 14,15- or 8,9-DHET were purified from sera using protein-G affinity chromatography (Pierce Co.). The IgG bound to the protein G column was eluted with 50 mM glycine-HCl buffer, pH 2.5, and immediately neutralized with 0.5 M tris-HCl, pH 7.6. This procedure did not affect the specificity of the antibodies.

Immunoaffinity Column Chromatography

Reacti-gel was obtained from Pierce Co. and anti-14,15-DHET immunoaffinity column was prepared according to the manufacturer's instruction. Capacity of the immunoaffinity column was measured by applying increasing amounts of 14,15-DHET (FIG. 1). Bound 14,15-DHET was washed with phosphate-buffered saline (PBS), pH 7.4, twice and eluted with acetonitrile:water (1:3), followed by acetone:water (90:10) and quantitated by ELISA. Capacity of the column was ˜0.5 μg 14,15-DHET/100 μl.

Conjugation of 14,15- or 8,9-DHETs to Horseradish Peroxidase (HRP)

Synthetic 14,15- or 8,9-DHETs were coupled to HRP using dicyclohexylcarbodiimide as previously described (25).

Solid Phase Competitive Enzyme-Linked Immunosorbent Assay (ELISA)

High-binding microplates were coated with protein G-purified IgG suspended in 1M carbonate, pH 9.0, (1 μg/well; 200 μl/well final volume) and then covered with parafilm. After overnight incubation at room temperature, the wells were gently washed five times with tris buffered saline (TBS), pH 7.5, containing 0.1% tween. Non-specific sites were blocked by the addition of 0.2 ml of 5% (w/v) nonfat dry milk in TBS. After two hours of incubation at room temperature, they were washed three times with TBS-tween. One μg/well of purified IgG was sufficient for quantitation of 0.5 to 100 μg/well of 14,15- or 8,9-DHETs. Samples and standards (150 μL) were added and the plates incubated for 20 minutes, after which 15 ng of the DHET-HRP conjugate was added in 50 μL of TBS. Following incubation for one hour to permit competitive binding to bound antibody, unbound material was removed by thorough washing of the wells with TBS-tween, and 150 μL of a colorimetric substrate for HRP [3,3′,5,5′ tetramethylbenzidine (TMB) and hydrogen peroxide] (Sigma Co.) was added. The plate is then incubated for 20 minutes, the reaction stopped by addition of 75 μL of 1 N H₂SO₄, and the absorbance at 450 nm was determined using a microtiter plate reader. Under these assay conditions, the amount of color in a well is inversely proportional to the initial concentration of the sample or a standard ligand.

Animal Treatment

Male and female Sprague-Dawley (SD) rats (160-200 g) were purchased from Charles River Laboratories (Wilmington, Mass.) and housed in a controlled environment and fed standard laboratory chow for at least 3 days before use. Rats are treated i.p. with dexamethasone (DEX) (10 mg/kg/d, for 4 days) or corn oil (2 ml/kg/d, for 4 days). DEX was dissolved in corn oil.

Rat Urine Collection

Male SHR and WKY rats were purchased from Charles River Laboratories and housed in metabolic cages for up to 3 days and urine was collected over triphenylphosphine in 24 hour intervals. Aliquots were stored at −80° C.

Preparation of Microsomes

Microsomes were prepared from rat livers and kidneys as described previously (27,28). Microsomes were stored at −80° C. in 50 mM Tris acetate buffer, pH 7.4, containing 1 mM EDTA and 20% glycerol until use. Protein was assayed by the method of BCA protein assay (Pierce, Rockford, Ill.).

Enzyme Assays Using ¹⁴C Arachidonic Acid (AA)

AA epoxygenase activity of microsomes obtained from untreated and rats after treatment with corn oil or DEX was assayed as previously described (1). After incubation of the microsomes (1 mg protein/ml, final concentration), 0.05 M Tris-HCl buffer, pH 7.5, containing 0.15 m KCl, 10 mM MgC₁₂, 8 mM sodium isocitrate and 0.5 IU of isocitrate dehydrogenase and, incubated with ¹⁴C AA (1 μCi/μmol, 100 μM, final concentration) in the presence of 1 mM NADPH at 30° C. The reaction products were taken at five and ten minutes and extracted into ethyl ether containing 0.05% acetic acid. The AA oxygenase metabolites were separated by HPLC and quantitated by a radiomatic Flo-One β-Oetector.

Enzyme Assays Using ELISA

Microsomes (0.5 mg of protein/ml) in 0.05 M Tris-HCl buffer, pH 7.5, containing 0.15 M KCl, 10 mM MgCl₂ and 100 μM AA (total volume, 0.5 ml) were incubated at 35° C. with and without 1 mM NADPH. The EET metabolites were hydrated to DHETs by addition of 20 μl acetic acid and incubated at room temperature for 18 hours. DHETs were extracted with 0.5 ml ethyl acetate and dried under nitrogen. The dried DHETs were reconstituted with TBS and quantitated by ELISA.

Slot Blot Analyses

Slot blot analyses were carried out using alkaline phosphatase system. Visualization was accomplished by incubation with a mixture of 5-bromo-4-chloro-3-indolylphosphatate p-toluidine and nitrobluetetrazolium.

Western Blot Analysis

SDS-PAGE was carried out on 10% acrylamide gel. The separated proteins were electroblotted onto cellulose membrane and Western blot analyses were carried out using alkaline phosphatase system as previously described (29). Visualization of P450 bands was accomplished by incubation with a mixture of 5-bromo-4-chloro-3-indolylphosphatate p-toluidine and nitrobluetetrazolium.

Statistics

Statistical analysis was carried out using Statview 512 software (Brain Power, Inc., Calabasas, Calif.) and significance between groups was analyzed using one factor anova (Fisher's PLSD).

Example 1

Development of the Immunoassay

Sensitivity of Antibodies Produced Against 14.15- and 8.9-DHETs

The 2nd and 3rd bleeds of a goat immunized with 14,15-DHET-KLH conjugates showed high binding to 14,15-DHET-BSA conjugates. Whereas preimmune sera of the goat showed binding with 14,15-DHET conjugates at a basal, level (FIG. 2A). The 3rd and 4th bleeds of a rabbit immunized with 8,9-DHET-KLH conjugates showed high binding to 8,9-DHET-BSA conjugates. Whereas preimmune sera of the rabbit showed binding with 8,9-DHET conjugates at a basal level (FIG. 2B).

A typical standard graph for 14,15-DHET is presented in FIG. 5. The r² value for the fit of the data to an equation describing an inverse logarithmic relationship of free 14,15-DHET to B/Bo was usually higher than 0.96. The detection limits for 14,15- and 8,9-DHET with ELISA were ˜1 pg and 10 pg, respectively.

Specificity of Anti-14.15- and 8.9-DHETs

The specificity of the 14,15-DHET ELISA was investigated using authentic DHET and a panel of eicosanoids (Table 5) which, based on their structure, might be anticipated to compete with 14,15-DHET for binding to antibodies against 14,15-DHET. Anti-14,15-DHET did not cross-react with 5,6-, 8,9-, 11,12- or 14,15-EET, 5,6-DHET, 15(S)HETE, 5(s)15(S)DiHETE, arachidonic acid, Thromboxane B₂, PGE₂, PGF_(2a) or 6-keto-PGF_(1a). There was a slight cross-reaction with 8,9- and 11,12-DHET (3.3% for both). These result demonstrated that anti-14,15-DHET does not recognize EETs.

The specificity of the antibody developed against 14,15-DHET was further investigated utilizing slot blot analysis. The 14,15-DHET conjugated BSA, BSA alone and 8,9-DHET conjugated to BSA were blotted onto cellulose membrane. Slot blot analysis was carried out with anti-14,15-DHET. Though the same amount of protein is loaded to each lane (proteins were visualized by amido black staining), the antibody cross-reacted with 14,15-DHET conjugated BSA whereas the antibody failed to cross-react with 8,9-DHET which is structurally very similar to 14,15-DHET (FIG. 6). Anti-8,9-DHET cross-reacted with both 8,9- and 14,15-DHETs (FIG. 7, Panel A). Antibodies non-specifically binding to anti-14,15-DHET were absorbed by incubation of the IgG with anti-14,15-DHET immunoaffinity resin. The resultant IgG against 8,9-DHET was form-specific (FIG. 7, Panel B).

The specificity of the anti-14,15-DHET was also investigated utilizing an immunoaffinity chromatography. A 14,15-DHET immunoaffinity column was prepared and increasing amounts of synthetic 14,15-DHET were applied to the immunoaffinity column. Levels of 14,15-DHET bound to the column were by ELISA. The bound DHET levels were correlated with levels of 14,15-DHET applied to the column with a correlation coefficient (r²) of 0.97 (FIG. 8). This result demonstrated that, under this experimental condition, the antibody cross-linked to resin bound to 14,15-DHET proportional to the amount of 14,15-DHET applied onto the column.

Example 2

Conjugated Forms

Existence of a Conjugated Form of 14,15-DHET in Urine Specimens

³H 14,15-DHET (55,660 cpm) and³H 8,9-DHET (13,899 cpm) were extracted into an equal volume of ethyl acetate. Recovery rates of ³H 14,15- and ³H 8,9-DHETs after the ethyl acetate extraction were ˜100% (Table 3). However, 14,15-DHET levels in urine samples obtained from spontaneously hypertensive rats (SHR) decreased to the levels lower than ˜40% after ethyl acetate extraction (Tables 1 and 2; FIG. 1). This result demonstrated that 14,15-DHET exists in the urine as free and conjugated forms and antibodies produced against 14,15-DHETs recognize both free and conjugated forms.

Capability of the 14,15- and 8,9-DHET antibodies to cross-react with conjugated 14,15- and 8,9-DHETs was further demonstrated by immunoassays using 14,15- and 8,9-DHETs conjugated to bovine serum albumin via their carboxyl ends (FIG. 2, Panels A and B).

Free and Conjugated 14.15-DHET Levels in Urine Specimens of Hypertensive Rats

The levels of 14,15-DHET in urine samples obtained from hypertensive rats (SHR) were measured by ELISA without pretreatment. The 14,15-DHET levels in hypertensive rats urine specimens were ˜56-fold higher than those of normotensive rats (WKY). The values obtained by without pretreatment of urine specimens obtained from SHR were ˜2 to 6-fold higher than the levels of these measured by ELISA after ethyl acetate extraction of the urine specimens (Tables 1 and 2; FIG. 1). Levels of 14,15-DHET obtained after purification of urine using ethyl acetate extraction is a measurement of free (or ethyl acetate extractable) 14,15-DHET whereas levels of 14,15-DHET obtained by ELISA without purification of urine is a measurement of both free (ethyl acetate extractable) and conjugated (ethyl acetate unexctractable) DHET. Both free and conjugated forms also contain 14,15-DHET metabolites different from free and conjugated 14,15-DHETs but which retain immunoreactivity with anti-14,15-DHET.

Example 3

Measurement of AA Epoxygenase Activity Using ELISA

NADPH-independent (AA epoxygenase-independent) EET formation with hepatic microsomes was measured (FIG. 3). The AA epoxygenase-independent EET formation activity was higher in hepatic microsomes obtained from rats treated with dexamethasone. Measurement of EET levels using ELISA will measure both AA epoxygenase-dependent and AA epoxygenase-independent EET formation activities. Thus, subtracting the amounts of NADPH-independent EETs from total (NADPH-dependent+NADPH-independent) EETs after incubation of the enzyme with NADPH provides better assessment of catalytic activity of AA epoxygenases. This is contrary to the measurement of AA epoxygenase activity using ¹⁴CAA (Table 4) which measures only AA-epoxygenase-dependent EET formation.

EET formation after incubation of AA with microsomes in presence or absence of NADPH was estimated using 14,15-DHET ELISA after chemical hydrolysis of the EETs to DHETs as described in “MATERIALS AND METHODS, Enzyme Assays Using ELISA on page 16.”

Decrease of CYP2C23. a Primary AA Epoxygenase in Liver, by Treatment of Rats

Hepatic 3A levels increased after treatment of rats with dexamethasone (FIG. 4). Hepatic CYP2C23 protein level decreased dramatically after dexamethasone treatment of rats (FIG. 4). The decrease of CYP2C23 levels in liver coincided with dramatic decrease of AA epoxygenase activity as measured by ELISA by subtracting the amounts of NADPH-independent EETs from total (NADPH-dependent+NADPH-independent) EETs (FIG. 3) or measurement of AA epoxygenase activity using ¹⁴C AA (Table 4). This result showed that 2C23 is a predominant AA epoxygenase isoform in the rat liver and hepatic AA epoxygenase expression can be lowered by treatment of rats with glucocorticoids including dexamethasone.

Through out this application, various publications, including Unite States patents, are referenced by author and year and patents by number. Full citations for the publications are listed below. The disclosures of these publications and patents in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. The invention has been used is intended to be in the nature of words of description rather than of limitation.

Obviously, many modifications and variations of the present invention are possible in the light of the above teachings. It is, therefore, to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.

TABLE 1 Levels of 14,15-DHET in urine specimens obtained from spontaneous hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY) analyzed by ELISA. ELISA was carried out using horseradish peroxidase system and the bound enzyme conjugate was detected by the addition of 3,3′5,5′-tetra-methylbenzidine (TMB). Reaction was stopped with an acid stop solution and read at 450 nm. Optical density at 450 nm was converted to B/Bo × 100. B, absorbance at 450 nm and Bo, the maximum absorbance reading obtained in the absence of DHET. 14,15-DHET Levels B/Bo × Sample # 100/25 μL pg/25 μL pg/mL Mean pg/mL (Ratio) 1 58.7 10.36 414.5 (SHR) 532.2 (56) 2 55.4 16.25 649.9 3 90.7 0.13 5.3 (WKY) 9.5  (1) 4 83.7 0.34 13.7

TABLE 2 Levels of 14,15-DHET in urine specimens obtained from spontaneous hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY) analyzed after ethyl actate extraction by ELISA. Urine specimen (25 μL) was acidified and extracted with an equal volume of ethyl acetate. The extract was dried under N₂ and reconstituted with tris-buffered saline (TBS). ELISA was carried out using horseradish peroxidase system and the bound enzyme conjugate was detected by the addition of 3,3′5,5′-tetra-methylbenzidine (TMB). Reaction was stopped with an acid stop solution and read at 450 nm. Optical density at 450 nm was converted to B/Bo × 100. B, absorbance at 450 nm and Bo, the maximum absorbance reading obtained in the absence of DHET. 14,15-DHET Levels B/Bo × Sample # 100/25 μL pg/25 μL pg/mL Mean pg/mL (Ratio) 1 58.5 4.54 181.6 (SHR) 145.2 (7.3) 2 61.7 2.72 108.8 3 71.9 0.53  21.2 (WKY)  19.8 (1)   4 72.8 0.46  18.4

TABLE 3 Recovery of ³H DHETs spiked in human urine after solid or liquid phase extraction Human urine samples were spiked with ³H 14,15-DHET (55,660 cpm) or ³H 8,9-DHET (13,899 cpm) and acidified to pH 3.0 and extracted into an equal volume of ethyl acetate. Extracts and remaining fractions were counted by a liquid scintillation counter. Alternatively, the spiked urine samples were acidified to pH 3.0 and applied to C-18 solid phase extraction column. The column was first washed with 10% acetonitrile in water, pH 3.0, followed by washing with n-heptane. The bound DHETs were eluted with 1 mL ethyl acetate:heptane (1:1). Elution of DHETs with 1 mL ethyl acetate:heptane (1:1) was repeated until radioactivity of the eluates were low. [³H] 14,15-DHET [³H] 8,9-DHET Treatment CPM % CPM % None 55,660 100 13,899 100 C18 Solid Phase Extraction Flow-Through 1,241 22.3 260 1.9 ± ± ± ± 343 0.6 38 0.3 1st Elution 22,544 40.5 5,294 38.1 ± ± ± ± 784 1.4 1,403 10.1 2nd Elution 14,247 25.6 1,928 13.9 ± ± ± ± 951 1.7 465 3.3 3rd Elution 1,101 2.0 138 1.0 ± ± ± ± 73 0.1 34 0.2 Liquid Phase Extraction with Ethyl Acetate Extracts 56,690 101.9 13,155 94.6 ± ± ± ± 850 1.5 334 2.4 Remaining 5,342 9.6 852 6.1 Fractions ± ± ± ± 2,291 4.1 182 1.3

TABLE 4 Arachidonic acid (AA) oxygenase activity assays of hepatic microsomes using ¹⁴C AA AA epoxygenase activity of microsomes obtained from untreated and rats after treatment with corn oil or dexamethasone (DEX) was assayed with ¹⁴C AA (1 μCi/μmol, 100 μM, final concentration) in the presence of 1 mM NADPH at 30° C. The reaction products were taken at 5 and 10 min. and extracted into ethyl ether containing 0.05% acetic acid). The AA oxygenase metabolites were separated by HPLC and quantitated by a radiomatic Flo-One B-detector. 8,9-EET + Incubation Time 14,15-EET 11,12-EET 5,6-EET (min) 5 10 5 10 5 10 Untreated 164^(a) 317 170 493  61 292 Corn Oil 231  420 292 585 158 244 DEX ND^(b)  97 ND  73 ND ND ^(a)ng EET formed/mg protein ^(b)Not Detectable: Lower than 6.1 ng/mg protein

TABLE 5 Specificity of anti-14,15-DHET IgG. A listing of the panel of eicosanoids used to analyze the specificity of the 14,15-DHET ELISA. These eicosanoids were selected based on the similarity of their structure to 14,15-DHET, as being anticipated to potentially compete with 14,15-DHET for binding to anti-14,15-DHET and thus interfere with such assays. Eicosanoid % binding of control 14,15-DHET 100.00 8,9-DHET 3.30 11,12-DHET 3.30 14,15-EET 1.50 15(S)HETE 1.00 8,9-EET 0.40 5(s)15(S)DiHETE 0.20 11,12-EET 0.05 arachidonic acid 0.05 5,6-DHET 0.02 5,6-EET 0.02 Thromboxane B2 0.02 PGE2 <0.01 PGF2α <0.01 6-keto-PGF1α <0.01

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What is claimed is:
 1. A method for determining both arachidonic acid (AA) epoxygenases and epoxide hydrolases activity comprising: converting AA to epoxyeicosatienioc acids (EETs); then converting EETs to DHETs; and measuring the amounts of NADPH-independent dihydroxyeicosatrienoic acids (DHETs) and total NADPH-dependent and independent DHETs which provides an indirect measurement of NADPH-dependent formation from AA, which correlates to AA epoxygenases and epoxide Lydrolase presence.
 2. A method as defined in claim 1 further including assessing NADPH-dependent EET formation activity of AA epoxygenases by subtracting amounts of NADPH-independent EETs from total NADPH dependent and independent EETs.
 3. A method as defined in claim 2 wherein said method is an immunoassay and said assessing step of the said immunoassay includes subtracting amounts of NADPH-independent EETs from total EETs after incubation of the AA epoxygenases with NADPH followed by measurement of total and NADPH-independent EET levels using antibodies.
 4. A method as defined in claim 3 further including using antibodies against DHETs.
 5. A method as defined in claim 4 wherein said using step includes using antibodies which recognize conjugate-free and conjugated forms of EET. 